Vitamin D is a hormone involved in calcium absorption from the gut and mobilization of calcium from bone. Before it can function as a hormone, vitamin D undergoes two separate hydroxylation steps. It is first hydroxylated in the liver, at carbon 25, to generate a biologically inactive metabolite, 25-hydroxyvitamin D (25(OH)D), by the enzyme vitamin D 25-hydroxylase (25-OHase). 25(OH)D is further hydroxylated by mitochondria in the kidney to one of two metabolites: 1.alpha.,25-dihydroxyvitamin D.sub.3 (1.alpha.,25(OH).sub.2 D.sub.3), the active form of the vitamin, and 24R,25-dihydroxyvitamin D.sub.3.
The conversion of 25(OH)D to 24R,25-dihydroxyvitamin D.sub.3 is catalyzed by the enzyme 25-hydroxyvitamin D.sub.3 24-hydroxylase (24-OHase). The enzyme has been cloned and its cDNA expressed by Ohyama et al., FEBS Lett, 278: 195-198 (1991). The conversion of 25(OH)D to 1.alpha.,25(OH).sub.2 D.sub.3 is catalyzed by 25-hydroxyvitamin D-1.alpha.-hydroxylase, a renal cytochrome P450 enzyme of the vitamin D pathway (hereinafter referred to as 1.alpha.-OHase).
Expression of 1.alpha.-OHase activity is under tight hormonal control. 1.alpha.-OHase activity is stimulated in mammalian cell culture systems by parathyroid hormone (PTH), while 1.alpha.,25(OH).sub.2 D.sub.3 represses it (Trechsel et al, FEBS Lett. 1.35:115-118 (1981); Henry, J., Biol Chem 254: 2722-2729 (1979)). These regulatory responses are rapid and have been demonstrated to require de novo mRNA synthesis (Turner, Vitamin D; Basic and Clinical Aspects, Kumar, R. ed. (The Hague: Martinus Nijhoff: 1984)) indicating that they may occur at the transcriptional level.
Abnormalities in any step of vitamin D metabolism, from dietary deficiency through metabolic errors to end-organ resistance (i.e. mutations of the receptor for 1.alpha.,25(OH).sub.2 D.sub.3) can result in rickets or osteomalacia. The first identified inborn defect in vitamin D metabolism was pseudovitamin D-deficiency rickets (PDDR). PDDR is an autosomal recessive disorder characterized at the biochemical level by low serum calcium, secondary hyperparathyroidism and early onset of rickets. PDDR appears to be caused by impaired activity of 1.alpha.-OHase.
The disease locus for PDDR was mapped by linkage analysis to 12q13-q14 by Labuda et al, Am J. Hum. Genet. 47:28-36 (1990), but the molecular defect underlying the enzymc dysfunction has remained elusive owing to the lack of sequence information for the gene coding for 1.alpha.-OHase and the consequent inability to produce clones of the gene.
Accordingly, a need exists for determination of the sequence of the polynucleotide coding for 1.alpha.-OHase and for clones of the 1.alpha. OHase cDNA.